This proposal will examine the hypothesis that the insulin resistance associated with obesity is a consequence of increased diacylglycerol (DAG) synthesis, with resultant elevation of protein kinase C (PKC) activity and feedback inhibition of insulin targets, such as the insulin receptor tyrosine kinase and glycogen synthase. The hypothesis is based on preliminary findings of a paradoxical increase in insulin-stimulated DAG synthesis and elevated PKC activity in two "insulin resistant" conditions, hyperinsulinemia/hyperglycemia and denervation. The increases in DAG under those conditions were glucose- and insulin-dependent, and occurred in a metabolically species of DAG. The increase in DAG was correlated to increased membrane-associated PKC activity. PKC has been reported to inhibit insulin action at various levels including the stimulation of the insulin-receptor tyrosine kinase, and glycogen synthase. Since DAG is an important signalling molecule and a key intermediate in the conversion of glucose to storage fat, its potential role as a link between obesity and insulin resistance will be examined. Funding is requested to initiate the proposed study to determine whether a lesion in enhanced DAG synthesis and PKC activity, similar to that caused by hyperglycemia/hyperinsulinemia and denervation, occurs in the adipose tissue of an obese animal model. The ultimate aim is to extended the study to define the biochemistry of insulin resistance in various forms of human obesity. The specific aims of this study are as follows: 1) The synthesis of DAG and other lipids, and analysis of their molecular subspecies, in adipocytes from obese animals will be compared with those from their lean littermates. Parallel studies to assess insulin sensitivity and action on insulin-receptor activity, glucose uptake and glycogen synthesis will be carried out. 2) The amounts of total and membrane-associated PKC in tissues under various test conditions will be measured by enzymatic and immunoblotting techniques to assess PKC activity. 3) The relationship between PKC activity and a) the insulin receptor, b) glucose transporters and c) glycogen synthesis and their activation by insulin will then be studied by enzymatic, metabolic labelling and immunoblotting techniques. These studies should determine whether obesity is accompanied by an alteration in the content and synthesis of specific pools of DAG and in PKC signalling in the adipose tissue. If so, a common biochemical mechanism by be responsible for insulin resistance of various peripheral tissues caused by obesity and other pathological conditions, and novel means to counteract the pathogenesis of NIDDM may be indicated.